Project Summary/Abstract Often called the ?first? hormone of reproduction, the hypothalamic peptide gonadotropin releasing hormone (GnRH) is the key and essential endocrine signal that activates the hypothalamic-pituitary-gonadal (HPG) axis and, thus, reproductive function in males and females. Released from axon terminals in the median eminence, GnRH is transported via the hypophyseal portal circulation to the anterior pituitary gland where it binds to high affinity GnRH receptors and stimulates the expression and release of the gonadotropins luteinizing hormone (LH) and follicle stimulating hormone (FSH). In females, an acute rise in LH is obligatory for inducing ovulation and as such is a mandatory event for reproduction and fertility. Over the last several decades much has been elucidated regarding the key cellular and biochemical events elicited by activation of the GnRH receptor including the identity of intracellular signaling intermediates that underlie changes in gonadotropin gene expression and secretion. Missing, however, has been the development and application of technologies that possess the spatial and temporal resolution necessary for analyzing immediate early events elicited by GnRH in living cells. Such events would include the formation of plasma membrane signaling domains. Recently, we have applied high resolution imaging to demonstrate GnRH-mediated production of reactive oxygen species (ROS) coupled to the opening of L-type calcium channels. Calcium and reactive oxygen species (ROS) are ubiquitous signaling molecules that influence cellular processes ranging from neurotransmitter release to apoptosis. The general goal of this proposal is to investigate the poorly understood mechanisms controlling calcium and ROS signaling mechanisms in pituitary gonadotropes. More specifically, this research investigates a novel regulatory mechanism where localized oxidant and calcium signaling microdomains functionally converge in gonadotropes following stimulation of the GnRH receptor. This promotes increased ROS generation and L-type calcium channel activity, increased calcium within the cells, and ultimately changes in gene expression required for ovulation. In this application we propose to test a model where the convergence of calcium and oxidant microdomain signaling is coupled to activation of ERK signaling. We will also investigate the underlying mechanisms regulating this signaling modality. Specific Aim 1 tests the hypothesis that subplasmalemmal GnRH-induced calcium and ROS microdomains colocalize and are functionally coupled. Specific Aim 2 tests the hypothesis that activated GnRH receptors assemble and form dynamic multi-protein signaling complexes necessary for transduction of colocalized calcium and ROS microdomains to ERK activation Specific Aim 3 tests the hypothesis that actin cytoskeletal dynamics are regulated oxidatively and are essential for GnRH receptor microdomain signaling. The experiments in these Specific Aims will use a combination of voltage-clamp electrophysiology, total internal reflection fluorescence (TIRF) microscopy, molecular biology, biochemistry, super resolution imaging, and proteomics to examine the structural and function of this local signaling mechanism. The outcome of these experiments will provide mechanistic insights into events underlying gonadotrope function and dysfunction and may lead to the development of new rational approaches for regulating GnRH receptor signaling with respect to fertility and other important health issues such as GnRH receptor-responsive carcinomas.